The central hypothesis of this proposal is that cysteine is a significant cause of oxidative stress without oxygen, and that reactive sulfur species (RSS) mediate this stress. Because some of the most notorious pathogens have a particular enzyme to maintain low intracellular cysteine, it is further proposed that cysteine homeostasis is a major factor in bacterial infections. We propose to study cysteine-mediated stress in the model bacterium Salmonella enterica. To test this hypothesis, direct evidence for RSS by electron paramagnetic resonsance (EPR) spectrosopy will be sought. Evidence for a likely effect of damage by RSS will be sought by examining the free sulfhydryl content of cells, and whether proteins have been cysteinylated. Evidence will be sought for the possibility that RSS are synergistic with reactive nitrogen species, which could account for the virulence of some pathogens. Finally, a gene profiling analysis will be performed to determine the responses to damage caused by high intracellular cysteine. Narrative: Although reactive sulfur species have been previously suggested as components of oxidative stress, the proposed studies could point to conditions that allow their study and establish their significance. The biochemistry of cysteine that is established with bacterial model systems could provide insight into other problems, such as heart disease, since homocysteine is an independent risk factor for heart disease, and the chemistry of cysteine- and homocysteine- mediated damage is likely to be similar. Finally, since a particular enzyme that degrades cysteine is found in a variety of successful pathogens, such as the agents of cholera, bubonic plague, and tuberculosis, control of intracellular cysteine is important for bacterial virulence.